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Title: Phosphoproteomic and signalling-protein interaction analyses downstream of the Erythropoietin receptor in erythroid progenitors
Author: Konstantinou-Tegou, Anastasios
ISNI:       0000 0001 3601 7515
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2008
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Erythropoietin holds an essential role in the process of red blood cell production. Upon binding to its receptor it activates a signalling pathway regulated mainly by tyrosinephosphorylation events. Despite the extensive study of Epo signalling in the past two decades, substantial questions sti11 remain open, regarding mainly the proteins involved in the signal transduction pathway. . In contrast to the majority of published Epo-related studies, which use mainly erythroid cancer cell lines or hematopoietic cel1s transfected with the EpoR, in this study we utilised immortalised murine erythroid progenitor cells in order to understand the intracellular effects of Epo stimulation. This cell system combines the characteristics and signa11ing networks ~f the primary erythroid cel1s, with the unlimited life span of the immortalised cell lines, thus constituting an important tool for large-scale analyses. By differential centrifugation a subcellular fraction was identified where the active EpoR . was found specifically localised. By immunoblotting and immunoprecipitation experiments the localisation and interactions of various signalling proteins were investigated . . .' in the same as well as other generated fractions. Additional1y, different approaches were designed and fol1owed for a more global analysis of Epo-induced effects in the cells. These included fluorescent two-dimensional electrophoresis, affinity chromatography purification of tyrosine phosphorylated proteins by the use of ·phosphotyrosine antibodies and recombinantly expressed SH2 domains, as well as isolation of phosphorylated peptides with titanium dioxide microcolumns. Following mass spectrometric analysis, a wide range of potential novel phosphorylated proteins was identified
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available